Antibody Composition For Prevention Or Treatment Of Mutant Hepatitis B Virus Infection

Abstract

The present invention provides an antibody that binds to the surface antigen (HBsAg) of hepatitis B virus (HBV) to neutralize the hepatitis B virus. The surface antigen-binding site of the antibody was found to play a very important role in viral replication, and when a mutation in the site occurs, viral replication is significantly inhibited, and thus at least HBV virus cannot cause a mutation in the site. In the present invention, it was confirmed by the use of patient-derived virus that the antibody of the present invention binds to either YMDD mutant hepatitis B virus, produced by conventional viral replication inhibitors, or G145R HBsAg mutants to which plasma-derived HBIG (hepatitis B immunoglobulin) does not bind. In addition, the in vivo effect of the antibody of the present invention was examined using chimpanzees which are unique animal models for hepatitis B virus. As a result, it was found that the antibody has the effect of neutralizing even wild-type hepatitis B virus in the in vivo model. Thus, it can be seen that the antibody of the present invention has the ability to bind not only to wild-type hepatitis B virus, but also mutant hepatitis B viruses having a polymerase YMDD mutant and a surface antigen G145R mutation, as well as various mutant viruses derived from patients. Thus, the antibody of the present invention can be effectively used for the prevention or treatment of infections with not only wild-type hepatitis B virus, but also mutant hepatitis B viruses.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This is a divisional under 35 U.S.C. 121 of U.S. patent application Ser. No. 14/412,138 filed Dec. 30, 2014 for AN ANTIBODY COMPOSITION FOR PREVENTION OR TREATMENT OF MUTANT HEPATITIS B VIRUS INFECTION, which in turn is a U.S. national phase under the provisions of 35 U.S.C. .sctn.371 of International Patent Application No. PCT/KR2013/006025 filed Jul. 8, 2013, which in turn claims priority of Korean Patent Application No. 10-2012-0075063 filed Jul. 10, 2012. The disclosures of all such patent applications are hereby incorporated herein by reference in their respective entireties, for all purposes.

TECHNICAL FIELD

[0002] The present invention relates to a composition for preventing or treating a disease caused by mutant hepatitis B virus, which contains, as an active ingredient, a neutralizing antibody against mutant human hepatitis B virus (HBV) to which a conventional viral replication inhibitor (e.g., lamivudine or adefovir dipivoxil) or a plasma-derived HBIG (hepatitis B immunoglobulin) does not work or bind.

BACKGROUND ART

[0003] Hepatitis B virus (HBV) is a virus with a DNA genome, which belongs to the Hepadnaviridae family and causes acute and chronic hepatitis. Hepatitis B virus (HBV) is classified into eight genotypes having a difference of about 8% or more in the gene nucleotide sequence, or it is classified into four serotypes adw, adr, ayw and ayr) based on the two antigenic determinants (d/y and w/r) of hepatitis B surface antigen (HBsAg). About 3.5 hundred million people worldwide have chronic hepatitis B virus (HBV) infection, and particularly, in Korea and China, people with chronic hepatitis B virus infection reach about 5-8%, and hepatitis B virus (HBV) infection is the major cause of liver disease and liver cancer. Currently developed vaccines can be somewhat effective in the prevention of hepatitis B virus infection, but a significant number of patients with chronic infection with hepatitis B virus still exist. Chronic infection with hepatitis B virus (HBV) causes hepatitis, cirrhosis and liver cancer, and the incidence of liver cancer is about 300 times higher in people with chronic hepatitis B virus than non-infected people. According to the WV-10 report, about 80% of liverance is caused by chronic hepatitis B.

[0004] Currently known therapeutic agents for hepatitis B include the nucleoside analogues including lamivudine and adefovir dipivoxil, which inhibit the DNA replication of hepatitis B virus (HBV) by inhibiting the reverse transcriptase of hepatitis B virus polymerase (HBV polymerase). However, when these drugs are administered for 3 years, drug-resistant virus occurs in about 75% of the patients to reduce the therapeutic effect of the drug. Due to this problem, it is impossible treat hepatitis B infection using the viral replication inhibitors alone. For this reason, it was attempted to use these inhibitors in combination with interferon agents, but these inhibitors are not currently used due to serious side effects.

[0005] For a similar purpose, a hepatitis B immune globulin (HBIG) preparation comprising a hepatitis B virus (HBV) antibody isolated from blood having a high antibody titer was considered. However, because the antibody of the HBIG preparation is isolated and purified from plasma, there are problems, including difficulty in obtaining plasma, the possibility of viral infection, low activity, high costs and the like.

[0006] In recent years, there have been reports of mutant viruses capable of avoiding such antibodies, for example, a mutant having a glycine-to-arginine substitution at position 145 of the surface protein of hepatitis B virus (HBV). In addition, various mutants capable of avoiding the antibodies have appeared. For this reason, it is difficult for the conventional hepatitis B virus therapeutic agents to show satisfactory therapeutic effects.

[0007] Thus, there is an urgent need to develop an antibody for treating hepatitis B virus (HBV), which binds specifically to a hepatitis B virus (HBV) epitope in which no mutation occurs, so that the therapeutic effect of the antibody is not reduced by the mutation.

DISCLOSURE OF INVENTION

[0008] It is an object of the present invention to provide a composition for preventing or treating a disease caused by infection with a mutant virus having resistance to a conventional therapeutic agent which has been used for the prevention or treatment of hepatitis B virus (hereinafter, referred to as "HBV").

[0009] To achieve the above object, the present invention provides an antibody composition for preventing or treating an infection with a HBV having a G145R mutation of HBV surface antigen (HBsAg) or an YMDD (tyrosine-methionine-aspartate-aspartate) mutation of HBV DNA polymerase (SEQ ID NO: 13), the composition comprising an antibody comprising:

[0010] a heavy-chain variable region having any one amino acid sequence selected from among SEQ ID NO: 1 to SEQ ID NO: 5; and a light-chain variable region having any one amino acid sequence selected from among SEQ ID NO: 6 to SEQ ID NO: 10.

[0011] Other features and embodiments of the present invention will be more apparent from the following detailed descriptions and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0013] FIG. 1 is a graphic diagram showing the HBV-neutralizing activity of the antibody of the present invention in chimpanzees.

[0014] FIG. 2 depicts photographs (see (a) and (c)) and a graphic diagram (see (b)), which show the results of immunoprecipitation assay performed to examine whether the antibody of the present invention binds to the HBV of the blood of hepatitis B patients.

[0015] In FIG. 2, (a), 1: 0.1 .mu.g of the antibody of the present invention, 2: 0.5 .mu.g of the antibody of the present invention, 3: 1 .mu.g of the antibody of the present invention, 4: 5 .mu.g of the antibody of the present invention, and 5: PBS buffer.

[0016] In FIG. 2, (c), 1: PBS, 2: treated with 1 .mu.g of anti-tetanus toxoid human antibody (TT-F9), 3: treated with 1 .mu.g of Hepabig, 4: treated with 1 .mu.g of anti-hepatitis B virus surface antigen humanized antibody (HuS 10), and 5: treated with 1 .mu.g of the antibody of the present invention.

[0017] FIG. 3 is a set of photographs showing the results of an immunohistochemical staining assay performed to examine whether antibodies bind to human liver tissue infected with HBV. Specifically, in FIG. 3, (a) is a photograph showing that the antibody of the present invention was strongly bound to HBV-infected human liver tissue, and (b) is a photograph showing isotype negative control antibody was not bound to the same tissue.

[0018] FIG. 4 is a genetic map of hepatitis B virus (HBV). The plasmid pHBV1.3-MBRI was constructed by inserting an 1.3-fold sequence of an HBV (adr subtype) gene (Gene Bank Accession No. DQ683578) (HBV gene from upstream of enhancer I of an HBV genome to downstream of a polyadenylation region) into the Pinel restriction enzyme site of pcDNA3.1 (Invitrogen, USA).

[0019] FIG. 5 shows the results of an experiment performed to examine the neutralizing activity of an antibody against G145R mutant virus using a hydrodynamic mouse model and indicates that the surface antigen and viral particles of wild-type HBV and G145R mutant HBV were all removed from mouse blood.

BEST MODE FOR CARRYING OUT THE INVENTION

[0020] Hereinafter, the present invention will be described in further detail.

[0021] The present invention is directed to an antibody composition for preventing or treating an infection with a HBV having a G145R mutation of HBV surface antigen (HBsAg) or a YMDD (tyrosine-methionine-aspartate-aspartate) mutation of HBV DNA polymerase, the composition comprising an antibody comprising:

[0022] a heavy-chain variable region having any one amino acid sequence selected from among SEQ ID NO: 1 to SEQ ID NO: 5; and a light-chain variable region having any one amino acid sequence selected from among SEQ ID NO: 6 to SEQ ID NO: 10.

[0023] The antibody according to the present invention may be an antibody against a HBV surface antigen (HBsAg) having a G145R mutation or a DNA polymerase YMDD motif mutation, produced from the cell line HBAb-49 (KCLRF-BP-00054). The G145R mutation is a glycine-to-arginine substitution at position 145 of HBV surface protein, to which plasma-derived HBIG does not bind, and the YMDD motif is located in the C end region of the DNA polymerase gene of hepatitis B virus and has a methionine (M)-to-valine (V) or isoleucine (I) substitution at position 552 of the amino acid sequence.

[0024] The antibody composition is used for the prevention or treatment of infection with mutant virus resistant to the HBV therapeutic agent lamivudine or adefovir dipivoxil.

[0025] In addition, the antibody composition may further comprise an antiviral agent. The antiviral agent is preferably one or more selected from among interferon, anti-HBV monoclonal antibodies, anti-HBV polyclonal antibodies, nucleoside analogues, DNA polymerase inhibitors, and siRNA preparations, but is not limited thereto.

[0026] The antibody composition preferably contains the antibody at a concentration of 0.1-50 mg/ml The present invention also provides a pharmaceutical formulation containing the antibody composition as an active ingredient. The pharmaceutical formulation is preferably administered to mammals including human at a dose of 0.001-10 mg/kg (bodyweight).

[0027] The pharmaceutical composition may be prepared into a pharmaceutical formulation in accordance with any conventional method. In preparation of the formulation, the antibody is preferably admixed or diluted with a carrier, or enclosed within a carrier. When the carrier is used as a diluent, it may be a solid, semi-solid or liquid material acting as a vehicle, excipient or medium for the active ingredient. Thus, the formulations may be in the form of a tablet, pill, powder, sachet, elixir, suspension, emulsion, solution, syrup, aerosol, soft and hard gelatin capsule, sterile injectable solution, sterile packaged powder and the like.

[0028] Examples of suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like. The compositions of the invention may be formulated according to any method well known in the art so as to provide quick, sustained or delayed release of the active ingredient after their administration to a mammal

[0029] In an experiment performed to demonstrate the HBV neutralizing activity of the antibody of the present invention using chimpanzees, it was shown that the chimpanzees were not infected with HBV for one year after administration of a mixture of HBV and the antibody. In chimpanzees used in a control group, it was shown that the HBV virus particle and surface antigen were produced and an antibody against the HBV surface antigen was produced during the recovery stage (see FIG. 1).

[0030] In addition, it was shown by an immunoprecipitation assay that the antibody of the present invention had an excellent ability to bind to the HBV of patient blood (see FIG. 2). In addition, it was shown by an immunohistochemical staining assay that the antibody of the present invention did strongly bind to HBV-infected human liver tissue (see FIG. 3).

[0031] The antibody according to the present invention may have the ability to bind to and neutralize the HBsAg of antibody-resistant and antibody-escapable HBV which cannot be inhibited by a conventional viral replication inhibitor (lamivudine or adefovir dipivoxil) or plasma-derived HBIG. In an example of the present invention, the binding ability of the antibody was examined by an enzyme-linked immunosorbent assay (ELISA) using patient's blood containing YMDD mutant virus having an YMDD mutation on the reverse transcriptase of hepatitis B virus polymerase, which has resistance to viral replication inhibitors. As a result, it was shown that the antibody strongly bind to all the YMDD mutant viruses (see Table 5 and Table 6).

[0032] The biggest characteristic of the antibody of the present invention is its ability to bind to and neutralize a mutant having a glycine-to-arginine substitution at position 145 of HBV surface protein, which cannot be neutralized by plasma-derived HBIG. To verify this ability, mutant virus was produced using a hydrodynamic mouse model, and whether the antibody has the ability to neutralize the produced mutant virus was examined As a result, it was shown that HBsAg and HBV in the blood of the mouse model were all removed (see FIG. 5).

[0033] It was shown that the antibody of the present invention did bind to hepatitis B viruses (HBVs) of patients, which recurred after liver transplantation, and that the HBV viruses were all mutants having a glycine-to-arginine substitution at position 145 of HBV surface protein (see Table 8).

[0034] The above-described results suggest that the antibody of the present invention and a composition comprising the same can be effectively used for the prevention or treatment of infection with mutant HBV virus having resistance to conventional therapeutic agents. Particularly, it can be seen that the antibody and the composition can be very effectively used for the prevention or treatment of infection with G145R mutant HBV or YMDD motif mutant HBV.

EXAMPLES

[0035] Hereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to a person having ordinary skill in the art that these examples are illustrative purposes only and are not to be construed to limit the scope of the present invention.

Example 1

Experiment on HBV-Neutralizing Ability in Chimpanzees

[0036] In order to examine whether the antibody of the present invention has the ability to neutralize HBV in vivo, the following experiment was performed.

[0037] HBV 100 CID.sub.50 (50% chimpanzee infectious doses) obtained from the Hepatitis Research Foundation (USA) was placed in three tubes. The antibody of the present invention comprising a heavy-chain variable region having amino acid sequence of SEQ ID NO: 2 and a light-chain variable region having amino acid sequence of SEQ ID NO: 7 was added to two of the three tubes in amounts of 0.1 mg and 10 mg, respectively, and no antibody was added to the remaining one tube. The mixture in each of the tubes was adjusted to a volume of 3 ml with PBS (phosphate buffered saline) buffer, after which the mixture was allowed to react at 37.degree. C. for 1 hour, and then at 4.degree. C. overnight, followed by freezing with liquid nitrogen, thereby preparing test materials.

[0038] For an animal experiment, the test materials were administered intravenously to three chimpanzees, respectively, which have never been infected with HBV (see Table 1).

TABLE-US-00001 TABLE 1 Dose of antibody administered to each chimpanzee Age Weight Dose of Sex (years) (kg) antibody Controls Chimpanzee 1 Male 4 12.2 -- Test group 1 Chimpanzee 2 Male 4 11.6 0.1 mg Test group 2 Chimpanzee 3 Female 4 10.8 10 mg

[0039] At 1-week intervals during a period ranging from 1 week after antibody administration to 8 weeks after antibody administration and at 2-week intervals after antibody administration, blood was collected from the chimpanzees to measure HBV infection-related indices, including HBV DNA, HBsAg (HBV surface antigen), anti-HBs (HBV surface antigen antibody), anti-HBc (HBV core antibody), ALT, AST and the like. In addition, the in vivo safety of the antibody was analyzed by blood and urine examinations.

[0040] In addition, the changes in the HBV DNA, HBsAg and anti-HBs of chimpanzee were measured, and the results of the measurement are graphically shown in FIG. 1.

[0041] As shown in Tables 2, 3 and 4, HBV infection was observed in chimpanzee 1 as the control, whereas no HBV infection was observed in chimpanzees 2 and 3, administered with antibody together with HBV, throughout the experimental period. Such results revealed that the antibody of the present invention has an excellent ability to neutralize HBV. In addition, no special abnormal findings were observed in liver function examination, various hematological examinations, urine examination and the like, suggesting that the antibody is safe in vivo.

TABLE-US-00002 TABLE 2 Measurement of HBV infection indices (chimpanzee 1) HBV PCR Time from ALT AST Log.sub.10 (DNA HBsAG Anti-HBs Anti-HBc administration (sf units) (sf units) mol/ml) (EIA) (EIA) (EIA) Before 1 week 6 11 N Admin. day 5 10 N After 1 week 15 13 N After 2 weeks 6 16 N After 3 weeks 8 22 N After 4 weeks 2 6 N After 5 weeks 6 11 N After 6 weeks 5 12 N After 7 weeks 6 20 N After 8 weeks 7 18 N After 10 weeks 8 18 2.21 .015(-) After 12 weeks 10 23 2.43 .023(-) After 14 weeks 13 25 3.24 .064(-) After 16 weeks 12 18 3.47 .209(+) After 18 weeks 8 20 4.10 .600(+) 1.004(-) After 20 weeks 8 13 4.50 >2.000(+) 1.264(-) After 22 weeks 10 12 4.82 >2.000(+) .056(-) 1.038(-) After 24 weeks 15 18 N 0.03(-) .085(-) .0129(+) After 28 weeks 23 22 N N >2.000(+) 0.156(+) After 32 weeks 19 18 N N >2.000(+) 0.119(+) After 36 weeks 21 19 N N >2.000(+) 0.061(+) After 40 weeks 7 23 N After 44 weeks 25, 24 19 N After 48 weeks 19 16 N After 51 weeks 28, 29 23 N

TABLE-US-00003 TABLE 3 Measurement of HBV infection indices (chimpanzee 2) ALT AST HBV PCR Anti- Anti- Time from (sf (sf Log.sub.10 (DNA HBs HBc administration units) units) mol/ml) (EIA) (EIA Before 1 week 26 22 N Admin. day 9 26, 25 N After 1 week 10 23 N After 2 weeks 6 24 N After 3 weeks 9 25 N After 4 weeks 4 18 N (-) After 5 weeks 9 37, 37 N (-) After 6 weeks 5 25 N (-) After 7 weeks 5 9 N (-) After 8 weeks 5 13 N (-) After 10 weeks 8 17 N After 12 weeks 14 21 N After 14 weeks 17 23 N After 16 weeks 15 19 N After 18 weeks 22 16 N After 20 weeks 20 16 N After 22 weeks 13 19 N After 24 weeks 24 22 N After 28 weeks 28, 28 25 N After 32 weeks 24 26 N After 36 weeks 23 25 N After 40 weeks 11 20 N After 44 weeks 27, 27 17 N After 48 weeks 18 13 N N After 51 weeks 30, 29 24 N N

TABLE-US-00004 TABLE 4 Measurement of HBV infection indices (chimpanzee 3) ALT AST HBV PCR Anti- Anti- Time from (sf (sf Log.sub.10 (DNA HBsAG HBs HBc administration units) units) mol/ml) (EIA) (EIA) (EIA) Before 1 week 5 18 N Admin. day 11 22 N After 1 week 7 18 N After 2 weeks 5 20 N After 3 weeks 13 31, 31 N After 4 weeks 9 19 N (-) (-) After 5 weeks 8 23 N (-) (-) After 6 weeks 14 26 N (-) (-) After 7 weeks 7 15 N (-) (-) After 8 weeks 10 19 N (-) (-) After 10 weeks 20 16 N After 12 weeks 13 19 N After 14 weeks 16 21 N After 16 weeks 16 24 2.24*, N, N After 18 weeks 21 24 N After 20 weeks 14 22 N After 22 weeks 16 19 N After 24 weeks 21 17 N After 28 weeks 18 21 N After 32 weeks 23 16 N After 36 weeks 22 17 N After 40 weeks 16 23 N After 44 weeks 24, 25 15 2.24*, N, N After 48 weeks 20 19 N N After 51 weeks 28, 31 22 N N (*borderline (+))

Example 2

Examination of HBV-Binding Ability of Antibody by Immunoprecipitation

[0042] Whether the antibody of the present invention comprising heavy-chain variable region having amino acid sequence of SEQ ID NO: 2 and a light-chain variable region having amino acid sequence of SEQ ID NO: 7 binds to HBV in hepatitis B patient blood (provided from Ajou University School of Medicine) was examined by immunoprecipitation (see FIG. 2).

[0043] (1) Preparation of Hepatitis B Patient Blood

[0044] 1,000 .mu.l of a 10-fold dilution of hepatitis B patient blood in 0.2% BSA/PBS buffer was allowed to react with a goat anti-human IgG (Fc specific)-agarose conjugate (Research Diagnostics Inc., Flanders, N.J.) to remove immunoglobulin from the blood.

[0045] (2) Binding Reaction Between Antibody and Goat Anti-Human IgG-Agarose Conjugate

[0046] 10 .mu.l of the antibody of the present invention (0.1, 0.5, 1 and 5 .mu.g), PBS solution and 50 .mu.l of a goat anti-human IgG-agarose conjugate (Research Diagnostics) were mixed with each other and allowed to react with stirring at room temperature for 1 hour, and then 10 mg of human immunoglobulin (I.V.-Globulin-S, Green Cross) was added thereto and allowed to react with stirring at room temperature for 1 hour so as to block the binding portion of the goat anti-human IgG-agarose conjugate. For comparison, 1 .mu.g of each of blood HBV antibody (Hepabig), TT-F9 (anti-tetanus toxoid human antibody) and HuS 10 (anti-hepatitis B virus surface antigen humanized antibody) was used in the same manner as above.

[0047] (3) Binding Reaction Between Antibody-Bound Goat Anti-Human IgG-Agarose Conjugate and Patient Blood

[0048] 200 .mu.l of the blood prepared in Example 2-(1) was mixed with the antibody-bound goat anti-human IgG-agarose conjugate prepared in Example 2-(2), and the mixture was stirred at room temperature for 1 hour to allow the antibody to react with the HBV of the patient blood.

[0049] (4) Examination of Precipitation of HBV

[0050] The reaction solution of Example 2-(3) was centrifuged, and the supernatant was collected and HBV in the supernatant was measured using a Cobas Amplicor HBV Monitor Test (v2.0; Roche Diagnostics, Basel, Switzerland).

[0051] The agarose remaining after centrifugation was washed 10 times with 0.2% BSA/PBS buffer, and then added to 100 .mu.l of the same buffer, and 5 .mu.l of 10% SDS, 2 .mu.l of 50 mM EDTA and 200 .mu.g of protease K (Sigma-Aldrich) were added thereto and allowed to react at 55.degree. C. for 30 minutes. Then, the supernatant was collected and DNA was isolated therefrom using a QIAquick PCR purification kit (Qiagen, Hilden, Germany), after which HBV-specific DNA was amplified by PCR using a LiquiMix GM PCR premix (Neurotics, Korea), primer M3 (SEQ ID NO: 11) and primer POL8 (SEQ ID NO: 12). Herein, the PCR was performed under the following conditions: initial denaturation at 55.degree. C. for 5 minutes, and then 35 cycles of 1 min at 95.degree. C., 1 min at 55.degree. C. and 1 min at 72.degree. C., followed by final extension at 72.degree. C. for 10 min. The amplified DNA was analyzed on 1.0% agarose gel. As controls, HBV humanized antibody and tetanus toxoid human antibody (Green Cross, Korea) were used. The results of the analysis are shown in FIG. 2.

[0052] As shown in FIG. 2, (a) and (b), the amount of precipitation of HBV increased as the amount of antibody used in the immunoprecipitation reaction increased, and the amount of HBV in the supernatant after the immunoprecipitation reaction increased as the amount of the antibody decreased. Also, the amount of precipitation of HBV increased as the amount of the antibody increased. In addition, as shown in FIG. 2, (c), when the same amount of the antibody was used, the HBV antibody (Hepabig) purified from blood did not precipitate HBV due to its low ability to bind to HBV, whereas the antibody of the present invention did precipitate HBV due to its high ability to bind to HBV.

Example 3

Examination of HBV-Binding Ability of Antibody by Immnunohistochemistry

[0053] Whether the antibody of the present invention comprising heavy-chain variable region having amino acid sequence of SEQ ID NO: 2 and a light-chain variable region having amino acid sequence of SEQ ID NO: 7 binds to HBV-infected tissue was examined by immnunohistochemistry.

[0054] A frozen slide having HBV-infected human liver tissue (Spring Bioscience, Fremont, Calif., USA, Catalog No. STS-025) was fixed with acetone and allowed to react with a dilution of hydrogen peroxide in methanol. Then, the tissue slide was allowed to react with normal rabbit serum, followed by sequential reactions with avidin and biotin. Then, the tissue slide was allowed to react with each of the antibody of the present invention and an isotype human immunoglobulin (IgG1 isotype negative control antibody; Sigma-Aldrich), which were biotinylated using an immunoprobe biotinylation kit (Sigma-Aldrich), and the tissue slide was allowed to react with StreptABComplex/HRP (Dako, Holland). Each of the reaction products was stained with 3,3'-diaminobenzidine tetrahydrochloride (DAB) and counterstained with haematoxylin, and the results of the staining are shown in FIG. 3, (a) and (b).

[0055] As can be seen in FIG. 3, (a) and (b), the isotype negative control antibody (see (b)) did not bind to the HBV-infected human liver tissue, whereas the antibody of the present invention (see (a)) did strongly bind to the HBV-infected human liver tissue.

Example 4

Examination of the Ability to Bind to HBV Replication Inhibitor-Resistant Mutant

[0056] In order to examine whether the antibody of the present invention comprising heavy-chain variable region having amino acid sequence of SEQ ID NO: 2 and a light-chain variable region having amino acid sequence of SEQ ID NO: 7 binds, patient blood samples (provided from St. Mary's Hospital, Catholic University) were allowed to react in a 96-welt plate coated with the antibody of the present invention, and detection was performed using a sheep anti-HBsAg/peroxidase conjugate in a Genedia HBsAg ELISA 3.0 kit (Green Cross MS, Korea). As a result, as shown in Table 5 below, the antibody of the present invention did strongly bind to the HBsAg of all YMDD mutant viruses. Thus, as can be seen in Table 5, the antibody of the present invention can bind to YMDD mutant virus in the blood of chronic hepatitis B (CHB) patients.

TABLE-US-00005 TABLE 5 Results of enzyme-linked immunosorbent assay (ELISA) for the ability of the antibody of the present invention to bind to YMDD mutant virus Sample A450 (-) Control 0.063 0.09 0.058 (+) Control 0.488 0.524 YMDD #1 1.16 YMDD #3 0.957 YMDD #4 1.019 YMDD #5 0.356 YMDD #6 1.043 YMDD #7 1.104 YMDD #8 1.143 YMDD #9 0.834 YMDD #10 1.134 YMDD #11 0.786 YMDD #12 0.876 YMDD #13 1.066 YMDD #14 0.815 YMW(+) 0.747 CSY(+) 1.023 SYW(-) 0.073 Q101K, 1126N, 0.857 G145A BSA(-) 0.251 0.263

Example 5

Examination of the Ability to Bind to Various HBsAg Mutants Derived from Chronic Hepatitis B, Liver Cirrhosis and Hepatocellular Carcinoma

[0057] Virus surface antigen (HBsAg) mutants derived from 100 chronic hepatitis B (CHB) patients, 100 liver cirrhosis (LC) patients and 100 hepatocellular carcinoma (HCC) patients were analyzed to examine whether the antibody of the present invention comprising heavy-chain variable region having amino acid sequence of SEQ ID NO: 2 and a light-chain variable region having amino acid sequence of SEQ ID NO: 7 bind to all the mutant viruses. Patient blood samples (provided from St. Mary's Hospital, Catholic University) were allowed to react in a 96-well plate coated with the antibody of the present invention, and detection was performed using a sheep anti-HBsAg/peroxidase conjugate in a Genedia HBsAg ELISA 3.0 kit (Green Cross MS, Korea). As a result, as shown in Table 6 below, the antibody of the present invention did strongly bind to all the HBsAg mutants derived from the patients.

TABLE-US-00006 TABLE 6 Results of measurement of binding of the antibody of the present invention to typical surface antigen mutant viruses Patient S mutation number ELISA NDA titer (amino acid 124-147) CH 15 3.059 3.864*10.sup.3 L110M, T113S, S114T, L126T, G130D, T131D, S143T, R160K CH 32 2.949 10.sup.8 P142T CH 33 2.833 10.sup.8 L126S CH 34 1.37 56646*10.sup.3 Y100S, L126S, T131N, M133T CH 62 3.085 10.sup.8 T131L, R160K CH 75 2.933 10.sup.8 L126S, T131N, M133T LC 32 2.726 <2.5 pcr P127R, Q129K, T131A, M133L, T140S, K141R, P142S, C147Y, A159W LC 53 3.497 <2.5 pcr L126T LC 59 3.633 <2.5 pcr T131P LC 98 3.553 9761*10.sup.3 G130N HCC 1 3.611 11943*10.sup.3 Q101K, L126T HCC 11 3.358 >100000*10.sup.3 L126T, G130N, R160K HCC 22 3.517 270.8*10.sup.3 Y100C, L126T HCC 94 3.556 39687*10.sup.3 T123A, S143W [CH: (chronic hepatitis, 100 patients); LC: liver cirrhosis, 100 patients); HCC: (hepatocellular carcinoma, 100 patients)]

Example 6

Examination of In Vivo Effect of Antibody in Acute Hepatitis B-Induced Mice

[0058] In this Example, C57BL6 mice showing symptoms similar to acute hepatitis B were made by injecting HBV DNA into mice by hydrodynamic injection, and the ability of the antibody of the present invention comprising heavy-chain variable region having amino acid sequence of SEQ ID NO: 2 and a light-chain variable region having amino acid sequence of SEQ ID NO: 7 to neutralize hepatitis B surface antigen (HBsAg) was measured.

[0059] The C57BL6 mice used were twenty 6-week-old female mice (weight: about 20 g; purchased from Charles Liver Laboratory, MA, USA) and divided into 4 groups, each consisting of 5 mice, as shown in Table 7 below. 20 .mu.g of a pHBV-MBRI vector (Shin et al., Virus Research 119, 146-153, 2006; see FIG. 4) obtained by inserting a HBV DNA nucleotide sequence into pcDNA3.1 (Invitrogen, USA) was diluted to a volume corresponding to 9.5% of the mouse weight and was injected into the tail vein of each of the mice at a rate of 0.3 ml/min to induce acute hepatitis B in the mice. After 24 hours, 0.2 ml of the test material shown in Table 7 below was injected into the tail vein of each of the mice. Before injection of the test material (0 hr) and at 24 and 48 hours after injection, blood was collected from the mice, and serum was separated therefrom and diluted 10-fold with goat serum, after which the concentration of HBsAg in the blood was measured using Genedia HBsAg ELISA 3.0 (Green Cross MS, Korea).

TABLE-US-00007 TABLE 7 Experimental design for measuring the ability to neutralize hepatitis B surface antigen (HBsAg) in mouse blood Group Number Test material and path Dose HBsAg (ayw) 5 PBS, intravenous 0.2 mL injection HBsAg (ayw) 5 rHBIG 0.1 mg (400 IU), 0.2 mL intravenous injection G145R 5 PBS, intravenous 0.2 mL injection G145R 5 rHBIG 0.1 mg (400 IU), 0.2 mL intravenous injection

[0060] The results of the measurement are shown in FIG. 5. As can be seen in FIG. 5, (a), in the control group injected intravenously with PBS among the wild-type virus groups, the blood HBsAg concentration and the HBV DNA replication were maintained at the peak levels up to 48 hours, whereas in the group administered with 0.1 mg of the antibody rHBIG, blood HBsAg and HBV DNA replication were not substantially detected after 24 hours up to 48 hours due to complete neutralization. In addition, as can be seen in FIG. 5, (b), in the control group administered intravenously with PBS among the G145R mutant virus groups, the blood HBsAg concentration and the HBV DNA were maintained at the peak levels up to 48 hours, whereas in the group administered with 0.1 mg of the antibody, blood HBsAg and HBV DNA replication were not substantially detected after 24 hours up to 48 hours due to complete neutralization. Thus, the above-described results indicate that the antibody of the present invention comprising heavy-chain variable region having amino acid sequence of SEQ ID NO: 2 and a light-chain variable region having amino acid sequence of SEQ ID NO: 7 has a very excellent neutralization effect against the wild-type and G145R mutant HBV surface antigens. In addition, the number of HBV DNA copies in each of the groups was quantified using real-time PCR, and as a result, viral DNA was detected in both the wild-type and G145R mutant HBVs. This suggests that the antibody of the present invention has a very excellent neutralization effect against both the wild-type and G145R mutant HBVs.

Example 7

Examination of the Ability to Bind to G145R HBsAg Mutants Derived from Patients in which HBV Recurred by G145R Mutants After Liver Transplantation

[0061] The ability of the antibody of the present invention comprising heavy-chain variable region having amino acid sequence of SEQ ID NO: 2 and a light-chain variable region having amino acid sequence of SEQ ID NO: 7 to bind to G145R HBsAg mutants derived from patients having a HBV which recurred by a G145R mutation in HBsAg was examined Patient blood samples were allowed to react in a 96-well plate coated with the antibody of the present invention, and detection was performed using a sheep anti-HBsAg/peroxidase conjugate in a Genedia HBsAg ELISA 3.0 kit (Green Cross MS, Korea). As a result, as can be seen in Table 8 below, the antibody did strongly bind to all the G145R HBsAg mutants.

TABLE-US-00008 TABLE 8 Results of measurement of binding of the antibody of the present invention to all HBsAg mutants derived from patients Immobilized antibody Sample rHBIG Mutation S** 2.526 G145R C** 2.471 G145R B** 3.078 G145R L** 2.717 G145R W** 2.660 G145R Negative 0.015 G145R control Positive 1.048 G145R control

INDUSTRIAL APPLICABILITY

[0062] As described above, the antibody composition of the present invention can be effectively used for the prevention or treatment of infection with mutant viruses having resistance to conventional therapeutic agents. Particularly, it can be very effectively used for the prevention or treatment of infection with G145R mutant HBV or YMDD motif mutant HBV.

[0063] Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Sequence CWU 1

1

131129PRTArtificialvariable region of human antibody H chain 1Gln Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Ser Leu Thr Lys Tyr 20 25 30 Lys Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Thr Ser Arg Asp Ile Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Val Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asp Gly Trp Leu Trp Gly Trp Asp Val Arg Ser Asn Tyr Tyr 100 105 110 Tyr Asn Ala Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 125 Ser 2129PRTArtificialvariable region of human antibody H chain 2Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Ser Leu Thr Lys Tyr 20 25 30 Lys Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Thr Ser Arg Asp Ile Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Val Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asp Gly Trp Leu Trp Gly Trp Asp Val Arg Ser Asn Tyr Tyr 100 105 110 Tyr Asn Ala Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 125 Ser 3117PRTArtificialvariable region of human antibody H chain 3Gln Val Gln Leu Val Gln Ser Gly Gly Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Leu Met Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asn Thr Tyr Ser Gly His Thr Asn Tyr Ala Arg Lys Phe 50 55 60 Arg Gly Arg Val Thr Met Thr Trp Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Pro Thr Trp Gly Ile Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 4117PRTArtificialvariable region of human antibody H chain 4Gln Val Gln Leu Val Gln Ser Gly Gly Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Leu Met Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asn Thr Tyr Ser Gly His Thr Asn Tyr Ala Arg Lys Phe 50 55 60 Arg Gly Arg Val Thr Met Thr Trp Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Pro Thr Trp Gly Ile Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 5117PRTArtificialvariable region of human antibody H chain 5Gln Val Gln Leu Val Gln Ser Gly Gly Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Leu Met Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asn Thr Tyr Ser Gly His Thr Asn Tyr Ala Arg Lys Phe 50 55 60 Arg Gly Arg Val Thr Met Thr Trp Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Pro Thr Trp Gly Ile Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 6108PRTArtificialvariable region of human antibody L chain 6Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Tyr Asn Ser 20 25 30 Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Ser Thr Ser Thr Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Thr Asn Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Val Thr Pro Glu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 7 108PRTArtificialvariable region of human antibody L chain 7Asp Ile Val Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Tyr Asn Ser 20 25 30 Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Ser Thr Ser Thr Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Thr Asn Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Val Thr Pro Glu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 8 107PRTArtificialvariable region of human antibody L chain 8Gln Ala Gly Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asp Asn Ile Gly Arg Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Thr Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Glu Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Val Val 85 90 95 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 9 108PRTArtificialvariable region of human antibody L chain 9Glu Ile Val Leu Thr Gln Ser Pro Pro Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn Asn Asn 20 25 30 Val Asn Trp Phe Gln Gln Glu Pro Gly Lys Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Ser Val Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys Arg 100 105 10114PRTArtificialvariable region of human antibody L chain 10Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Lys Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Gln Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ser 85 90 95 Thr Gln Phe Pro Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg 1125DNAArtificialprimer M3 11ctgggaggag ttgggggagg agatt 251219DNAArtificialprimer POL8 12aggatagaac ctagcaggc 1913832PRTArtificial SequenceYMDD mutation of HBV DNA polymerase 13Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp Asp 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Asp 20 25 30 Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Val 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Ser 50 55 60 Ser Thr Val Pro Val Phe Asn Pro His Trp Lys Thr Pro Ser Phe Pro 65 70 75 80 Asn Ile His Leu His Gln Asp Ile Ile Lys Lys Cys Glu Gln Phe Val 85 90 95 Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Gln Leu Ile Met Pro 100 105 110 Ala Arg Phe Tyr Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp Lys Gly 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu His Leu Val Asn His Tyr Phe Gln Thr 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Arg 145 150 155 160 Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Glu 165 170 175 Gln Glu Leu Gln His Gly Ala Glu Ser Phe His Gln Gln Ser Ser Gly 180 185 190 Ile Leu Ser Arg Pro Pro Val Gly Ser Ser Leu Gln Ser Lys His Arg 195 200 205 Lys Ser Arg Leu Gly Leu Gln Ser Gln Gln Gly His Leu Ala Arg Arg 210 215 220 Gln Gln Gly Arg Ser Trp Ser Ile Arg Ala Gly Ile His Pro Thr Ala 225 230 235 240 Arg Arg Pro Phe Gly Val Glu Pro Ser Gly Ser Gly His Asn Thr Asn 245 250 255 Leu Ala Ser Lys Ser Ala Ser Cys Ile Tyr Gln Ser Pro Val Arg Lys 260 265 270 Ala Ala Tyr Pro Ala Val Ser Thr Phe Glu Lys His Ser Ser Ser Gly 275 280 285 His Ala Val Glu Leu His Asn Phe Pro Pro Asn Ser Ala Arg Ser Gln 290 295 300 Gly Glu Arg Pro Val Phe Pro Cys Trp Trp Leu Gln Phe Arg Asn Ser 305 310 315 320 Lys Pro Cys Ser Asp Tyr Cys Leu Ser His Ile Val Asn Leu Leu Glu 325 330 335 Asp Trp Gly Pro Cys Thr Glu His Gly Glu His His Ile Arg Ile Pro 340 345 350 Arg Thr Pro Ala Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn 355 360 365 Pro His Asn Thr Ala Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe 370 375 380 Ser Arg Gly Asn His Arg Val Ser Trp Pro Lys Phe Ala Val Pro Asn 385 390 395 400 Leu Gln Ser Leu Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser 405 410 415 Leu Asp Val Ser Ala Ala Phe Tyr His Leu Pro Leu His Pro Ala Ser 420 425 430 Met Pro His Leu Leu Val Gly Ser Thr Gly Leu Ser Arg Tyr Val Ala 435 440 445 Arg Val Ser Ser Asn Ser Arg Ile Phe Asn His Gln Arg Gly Thr Met 450 455 460 Gln Asn Leu His Asp Tyr Cys Ser Arg Asn Leu Tyr Val Ser Leu Leu 465 470 475 480 Leu Leu Tyr Gln Thr Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro 485 490 495 Ile Ile Leu Gly Phe Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro 500 505 510 Phe Leu Leu Ala Gln Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg 515 520 525 Ala Phe Pro His Cys Leu Ala Phe Ser Tyr Met Asp Asp Val Val Leu 530 535 540 Gly Ala Lys Ser Val Gln His Leu Glu Ser Leu Phe Thr Ala Val Thr 545 550 555 560 Asn Phe Leu Leu Ser Leu Gly Ile His Leu Asn Pro Asn Lys Thr Lys 565 570 575 Arg Trp Gly Tyr Ser Leu His Phe Met Gly Tyr Val Ile Gly Ser Tyr 580 585 590 Gly Ser Leu Pro Gln Asp His Ile Ile Gln Lys Ile Lys Glu Cys Phe 595 600 605 Arg Lys Leu Pro Ile Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg 610 615 620 Ile Val Gly Leu Leu Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr 625 630 635 640 Pro Ala Leu Met Pro Leu Tyr Ala Cys Ile Gln Ser Lys Gln Ala Phe 645 650 655 Thr Phe Ser Pro Thr Tyr Lys Ala Phe Leu Cys Lys Gln Tyr Leu Asn 660 665 670 Leu Tyr Pro Val Ala Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Ala 675 680 685 Asp Ala Thr Pro Thr Gly Trp Gly Leu Val Met Gly His Gln Arg Met 690 695 700 Arg Gly Thr Phe Leu Ala Pro Leu Pro Ile His Thr Ala Glu Leu Leu 705 710 715 720 Ala Ala Cys Phe Ala Arg Ser Arg Ser Gly Ala Asn Ile Leu Gly Thr 725 730 735 Asp Asn Ser Val Val Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu 740 745 750 Leu Gly Cys Ala Ala Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr 755 760 765 Val Pro Ser Ala Leu Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu 770 775 780 Gly Ile Phe Arg Pro Leu Leu Arg Leu Pro Phe Arg Pro Thr Thr Gly 785 790 795 800 Arg Thr Ser Leu Tyr Ala Asp Ser Pro Ser Val Pro Ser His Leu Pro 805 810 815 Val Arg Val His Phe Ala Ser Pro Leu His Val Ala Trp Arg Pro Pro 820 825 830

Claims

1. An antibody composition for preventing or treating an infection with a HBV having a G145R mutation of HBV surface antigen (HBsAg) or a YMDD (tyrosine-methionine-aspartate-aspartate) mutation of HBV DNA polymerase, the composition comprising, as an active ingredient, an antibody comprising: a heavy-chain variable region (V.sub.H) having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3 and 5; and a light-chain variable region (V.sub.L) having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 8 and 10.

2. The antibody composition of claim 1, further comprising another antiviral agent.

3. The antibody composition of claim 2, wherein the antiviral agent comprises one or more selected from the group consisting of interferon, anti-HBV monoclonal antibodies, anti-HBV polyclonal antibodies, nucleoside analogues, DNA polymerase inhibitors, and siRNA preparations.

4. The antibody composition of claim 1, wherein the YMDD mutation is a M552V or M5521 mutation.

5. The antibody composition of claim 1, wherein the virus is mutant hepatitis B virus (HBV) resistant to lamivudine, adefovir dipivoxil, or HBIG(hepatitis B immunoglobulin).

6. The antibody composition of claim 1, wherein the antibody is contained at a concentration of 0.1-50 mg/ml.

7. A pharmaceutical formulation containing the antibody composition of claim 1 as an active ingredient.

8. The pharmaceutical formulation of claim 7, further comprising carriers, excipients, and/or diluents.

9. The pharmaceutical formulation of claim 7, wherein the formulation is in the form selected from the group consisting of a tablet, pill, powder, sachet, elixir, suspension, emulsion, solution, syrup, aerosol, soft and hard gelatin capsule, sterile injectable solution, and sterile packaged powder.

10. The pharmaceutical formulation of claim 7, wherein the pharmaceutical formulation is preferably administered to mammals at a dose of 0.001-10 mg/kg.

11. The pharmaceutical formulation of claim 7, further comprising another antiviral agent.

12. The pharmaceutical formulation of claim 11, wherein the antiviral agent comprises one or more selected from the group consisting of interferon, anti-HBV monoclonal antibodies, anti-HBV polyclonal antibodies, nucleoside analogues, DNA polymerase inhibitors, and siRNA preparations.

13. The pharmaceutical formulation of claim 7, wherein the YMDD mutation is a M552V or M5521 mutation.

14. The pharmaceutical formulation of claim 7, wherein the virus is mutant hepatitis B virus (HBV) resistant to lamivudine, adefovir dipivoxil, or HBIG(hepatitis B immunoglobulin).

15. The pharmaceutical formulation of claim 7, wherein the antibody is contained at a concentration of 0.1-50 mg/ml.